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Screening of Pro–Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes Hwa Kyoung Lee, Yujean Lee, Hyori Kim, Hye-Eun Lee, Hyejin Chang, Ki Tae Nam, Dae Hong Jeong, and Junho Chung ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 26 May 2017 Downloaded from http://pubs.acs.org on May 27, 2017

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ACS Synthetic Biology

Screening of Pro–Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes

Hwa Kyoung Lee†, ‡, ¶, Yujean Lee†, ||, Hyori Kimⱴ, Hye-Eun Lee§, Hyejin Chang#, Ki Tae Nam§, Dae Hong Jeong#, Junho Chung*, †, ‡, ¶ † Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea ‡ Cancer Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea ¶ Department of Biomedical Sciences, Seoul National University Graduate School, 101 Daehakro, Jongno-gu, Seoul, 03080, Republic of Korea § Department of Materials Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea # Department of Chemistry Education, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea. ⱴ Biomedical Research Center, Asan Institute for Life Sciences, Asan Medical Center 88, 43 Olympic-ro, Songpa-gu, Seoul, 05505, Republic of Korea

ACS Paragon Plus Environment

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ABSTRACT:

Bacteriophages are thought to be ideal vehicles for linking antibodies to

nanoparticles. Here, we define the

sequence

of

peptides

exposed as a fusion protein on M13 bacteriophages to yield optimal

binding

of

gold

nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X)2DP, AE(X)3DP, AE(X)4DP, AE(X)5DP, and AE(X)6DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X)2DP, AE(X)3DP, and AE(X)4DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro–Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala– Glu–Pro–Asp–Asp–Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

KEYWORDS: Antibody, bacteriophage, helper bacteriophage


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Antibodies are used widely for antigen detection in various assay formats including enzyme immunoassays, radioimmunoassays, and fluorescence immunoassays. For these assays, antibodies are labeled with diverse compounds including enzymes, radioisotopes, and fluorochromes. Various technologies for the simultaneous detection of multiple antigens are under active development. However, these multiplex assays inevitably require the labeling of antibodies with compounds that possess differentiable signals. Until now, the most frequently used approach has used fluorochromes that emit different wavelengths; however, because of its narrower bandwidth and equivalent sensitivity, the recently introduced surface-enhanced Raman scattering (SERS) technique provides superior multiplexing capacity compared with conventional fluorochromes.1, 2 Because normal Raman scattering generates a very weak signal intensity, noble metal nanoparticles are commonly used to amplify the Raman scattering signal. At present, various transformations in the shape3 of nanoparticles are also being pursued to optimize the amplifying signal intensity. Bacteriophages are considered ideal vehicles for linking antibody molecules to nanoparticles. The pIII minor capsid protein displays an antibody molecule on the phage surface that serves as a fusion protein.4 However, most of the bacteriophage surface is covered with the major capsid protein pVIII. Hence, when the exposed N-terminal amino acid is replaced with a suitable amino acid motif,5 pVIII can function as an anchor for nanoparticles.6 This system is more advantageous than other antibody conjugation methods because the packaging of bacteriophages can be achieved in an economical, high-throughput manner. In addition, because bacteriophages remain stable under various physicochemical stresses, including freezing and thawing,7 and exposure to low8 or high pH,9 nanoparticles conjugated bacteriophages can be applied in various conditions.


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Gold nanocubes are one type of gold nanoparticle produced by the cationic surfactant cetyltrimethylammonium bromide (CTAB). They have edge structures as well as interstitial junctions between the edges, which can improve the SERS-induced signal. A helper bacteriophage with the exposed pVIII N-terminal sequence of Val–Ser–Gly–Ser–Ser–Pro–Asp– Ser (VSGSSPDS) has been screened from a bacteriophage library with randomized pVIII Nterminal sequences based on its reactivity to gold films.10 Additionally, bacteriophages with the VSGSSPDS N-terminal sequence have been applied to immunoassays,11 the formation of gold nanowires,12 and colloidal nanoparticle separation.13 Unfortunately, when applying this bacteriophage system in our experiments, we found that the amplification yield and plaque size of the bacteriophages were significantly lower than those of the wild-type helper bacteriophage (VCSM13). There are a limited number of reports on the effects of the length and primary sequence of the exposed N-terminal amino acid motif on phage amplification. It is not clear whether a mutant bacteriophage can be amplified with an efficiency similar to that of the wild-type phage or exhibit reactivity to gold nanoparticles. To address these questions, we constructed a series of mutant pVIII bacteriophage libraries with randomized exposed N-terminal amino acid motifs in which the amino acid length varied from two to six residues. To introduce the artificial sequences at the exposed pVIII N-terminal, we generated a KpnI restriction site near the 5′ end of pVIII corresponding to the leader peptide by replacing cytosine (C) and thymine (T) with guanine (G) and adenine (A), respectively, at the 3199 and 3202 positions in the VCSM13 genome (Accession No. AY598820; Figure 1). Because these were all silent mutations, no changes in the primary sequence of the leader peptide were introduced. Subsequently, we generated five libraries that encompassed the gene from the KpnI restriction


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site at gVIII to the BamHI restriction site at gIII using a set of sense primers encoding (NNK)2, (NNK)3, (NNK)4, (NNK)5, and (NNK)6, which replaced the exposed pVIII Gly–Asp (GD) motif (Figure 1).

Figure 1. Modification of the VCSM13 bacteriophage genome and construction of randomized pVIII N-terminal exposed residue libraries. A restriction endonuclease recognition site (KpnI) was introduced into the sequence encoding the pVIII coat protein leader peptide (LV), using the SnaBI and BamHI restriction enzyme sites, which are located in the VCSM13 genome and indicated in bold letters. Two amino acids (GD) exposed at the N-terminal pVIII coat protein were replaced with random amino acids encoded by degenerate codons (N = A, T, G and C, and K = G and T), as indicated in the box.

After preparation of NNK VCSM13-pVIII randomized libraries in phagemid DNA form, we transformed them into E. coli ER2738 cells and obtained both double-strand phagemid DNA and single-stranded phage DNA (ssDNA). The prepared phagemid DNA and phage DNA were subjected to next-generation DNA sequencing (NGS) analysis (Macrogen, Seoul, Korea). The number of reads in each library was around 1 × 106 (Table 1). The percentage of clones with the


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right length and sequence varied from 69.9% to 98.7% among the libraries, and we concluded that the quality of the libraries was acceptable. We confirmed that the complexity of clones found in the phagemid and phage DNAs through NGS exceeded the theoretical complexity in the (NNK)2 and (NNK)3 libraries. In the (NNK)4, (NNK)5, and (NNK)6 libraries, the theoretical complexity was too high to be confirmed by NGS. Because the percentage of the unique clones among the clones with the right length and sequence varied from 48.4% to 91.4%, we also concluded that the complexity of these libraries was acceptable. Next, we determined the sequences of the phages that succeeded in forming plaques. Sequence analysis revealed that all eight plaques selected from the (NNK)6 library had the original wild-type helper bacteriophage sequence Ala–Glu–Gly–Asp–Asp–Pro (AEGDDP), which might have occurred as a result of contamination with wild-type bacteriophage. The results of the sequence analysis also revealed that eight plaques selected from the (NNK)5 library had the sequence Asp–Val–Ala–Gly–Ala (DVAGA). However, clones with diverse sequences were identified in the remaining three libraries (Figure 2).

Figure 2. The exposed N-terminal sequence obtained from plaques before and after three rounds of biopanning. After obtaining the bacteriophage library with randomized N-terminal pVIII,


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approximately 30 plaques were randomly selected before and after three rounds of biopanning for gold nanocubes. The N-terminal sequences and their frequencies are displayed.

Unexpectedly, although the phages in the culture supernatant were quite diverse, we obtained only one plaque-forming phage clone from the (NNK)5 library. There was a marked discrepancy between the diversity of phages in the culture supernatant and that of plaque-forming phages in other three libraries. This suggests that the artificial sequences may have hindered one or more critical steps in the phage-amplification process, such as phage particle assembly or infection, as described earlier,14 and that the degree of hindrance might be changed depending on the culture conditions. To select the phage clones with affinity to the gold nanocubes, the gold nanocubes were synthesized using the seed-mediated method as previously described.15 To generate a large enhancement of the electromagnetic field in nanostructures, it is essential to create nanoparticles with sharp edges or assemblies of nanoparticles that are in close proximity. Gold nanocubes contain sharp corners on each side; thus, the morphology itself generates an intensified electromagnetic field. Moreover, the assembly of nanoparticles with well-controlled gaps can be easily achieved because of the flat faces of the cubic nanoparticle. 16 The controlled interparticle gap results in uniform enhancement, which is an integral part of SERS applications. Based on these advantages of the cubic shape, we used 50 nm gold nanocubes to fabricate a SERS active nanostructure. The absorption spectrum of the synthesized gold nanocubes was measured at 532 nm, and the color of the solution changed from light brown to red after the synthesis (data not shown). Biopanning of the VCSM13-pVIII randomized libraries with exposed (NNK)2, (NNK)3, and (NNK)4 residues was performed against the synthesized gold nanocubes. After three rounds


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of biopanning, phage clones were selected from the output titer plate and used to infect E. coli ER2738 cells. Phagemid DNA was purified from the culture using a Qiagen Plasmid Mini Kit (Qiagen) according to the manufacturer’s guidelines. Within the (NNK)2 VCSM13-pVIII randomized library, 93.33% of the selected clones possessed the Pro–Asp (PD) sequence (Figure 2). The sequences of the clones selected from the (NNK)3 library were diverse, but clones encoding the Ser–Asp–Gly (SDG) sequence were the most prevalent. However, all of the clones enriched from the (NNK)4 library possessed the Ala–Gly–Asp–Ser (AGDS) sequence. The discovery that PD was the found at a frequency of 93.33 % from the (NNK)2 VCSM13-pVIII randomized library was unexpected and suggested that the PD phage either possessed an increased affinity for the gold nanocubes or was more efficient during amplification. Interestingly, we noted that the plaque size of the PD bacteriophages was similar to that of the wild-type bacteriophages, whereas the plaque size of the VSGSSPDS bacteriophages was significantly smaller (Figure 3A–D). The plaque size varies according to factors such as the virion size, adsorption rate, lysis time,17 and plating conditions.18 For example, increases in virion size typically correspond to decreases in plaque size.17 We attempted to discern whether plaque sizes indicated amplification efficiency of the bacteriophages. The amount of bacteriophages obtained from single plaque was determined by titration as previously described.19 This experiment was performed in quadruplicate, and the average quantity of wildtype, VSGSSPDS, and PD helper bacteriophages obtained was 2.5 × 1011, 7.8 × 109, and 1.9 × 1011 plaque-forming units (pfu)/mL, respectively (Figure 3E). These results indicated that the amplification efficiency of the PD helper bacteriophages was similar to that of wild-type bacteriophages, whereas the VSGSSPDS bacteriophages were less efficient. All of these observations suggested that the PD bacteriophage is the ideal vehicle to gold nanocubes.


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Figure 3. Plaque size and amplification efficiency of wild-type, VSGSSPDS-VCSM13, and PDVCSM13 bacteriophages. (A–C) Photographic images of plaques produced by (A) wild-type phages, (B) VSGSSPDS-VCSM13 phages, and (C) PD-VCSM13 phages. (D) The plaque diameters were measured and are expressed as the mean ± SD of quintuplicate measurements. ** P < 0.005. (E) Three VCSM13 bacteriophages were amplified overnight, isolated from the culture supernatant by polyethylene glycol precipitation, and titrated. The titers of the bacteriophages obtained are expressed as the mean ± SD of quadruplicate measurements. ** P < 0.005. pfu = plaque-forming units. To test whether the PD helper bacteriophages are compatible with the antibody fragment displayed at pIII, phage ELISA was performed. Prostate-specific antigen (PSA) is a well-known biomarker for prostate cancer. In most patients with prostate cancer, the PSA level in serum is


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elevated20. Therefore, detection of the PSA is commonly used to diagnose prostate cancer. The gene encoding PSA and anti-PSA scFv was subcloned into the modified pCEP4 vector, followed by the expression and purification of the recombinant PSA protein and anti-PSA scFv, as described previously.21, 22 Bacteriophages displaying anti-PSA scFv were amplified using wildtype, VSGSSPDS, and PD helper bacteriophages, as described previously.19 The bacteriophage that was amplified using the PD helper bacteriophage showed a signal compatible with that obtained using the wild-type helper bacteriophage (Figure 5). The bacteriophage that was amplified using the PD helper bacteriophage exhibited the coexistence of the antigen and gold nanocubes in our prior experiment.23 Scanning electron microscopy (SEM) was performed to confirm the binding mode of helper bacteriophages to gold nanocubes. Evaluation of the resulting images revealed that the wild-type helper bacteriophages and gold nanocubes formed large aggregates (Figure 4A, 4B and S1). The exposed pVIII portion on the surface of the wild-type helper bacteriophages generally possesses a negative charge.5 Hence, a likely explanation for the formation of wild-type helper bacteriophage and gold nanocube aggregates was the charge interaction between the negative charge on the bacteriophages and the positive charge on the CTAB carbon chain. When VSGSSPDS helper bacteriophages were mixed with gold nanocubes, we observed a large number of gold nanocubes hovering but not forming complexes with the phages in the solution (Figure 4C, 4D and S2). However, in the complexes comprising PD helper bacteriophages and gold nanocubes, the nanocubes dispersed evenly along the length of the filamentous bacteriophages and significantly fewer aggregates were apparent (Figure 4E ,4F, and 4G). In addition, only a few unbound gold nanocubes were observed in the background (Figure S3). Based on these observations, we conclude that the PD helper bacteriophages formed


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uniform complexes more efficiently and without aggregating through the hydrophobic interactions between the CTAB and proline.23 Currently, we cannot explain precisely why the substitution of Gly with Pro in the exposed portion of pVIII provided superior characteristics to the structure. Nevertheless, it is well known that a short peptide cannot form a rigid fixed structure. The presence of a proline residue might provide a uniform rigid kinked structure on every pVIII. This might explain why the PD bacteriophage formed uniform complexes without aggregation

Figure 4. SEM images of gold nanocubes bound to bacteriophages. Bacteriophages (2 × 10

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pfu/mL) were incubated with gold nanocubes (Abs 540 = 2.0, 1 mL) for 1 h at room temperature. SEM images were obtained from (A, B) wild-type bacteriophages, (C, D) VSGSSPDS bacteriophages, (E, F, G) PD bacteriophages, and (H) without bacteriophages. Gold nanocubes are indicated by white spots, and the unbound parts of the bacteriophages are indicated by the


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dark lines. (I) Raman spectra of 4-chlorobenzenethiol (4-CBT)-labeled gold nanocubes (black), and complexes of gold nanocubes and PD bacteriophages (red). The excitation laser line was 647 nm (58.8 mW) and the acquisition time for each measurement was 10 s. a.u. = arbitrary units.

To investigate the potential application in multiplex assays, we characterized the SERS signal enhanced by the formation of gold nanocube and bacteriophage complexes. The SERS signal was detected from the gold nanocubes complexed with PD helper bacteriophages (Figure 4I). The results shown in Figure 4I suggest that the SERS intensity obtained from complexes of gold nanocubes and PD helper bacteriophages was higher than that from the intensity of gold nanocubes alone. Because the 4-CBT-labeled gold nanocubes existed separately from each other in solution, there was a relatively weak local electromagnetic field around the gold nanocubes, which resulted in a weak SERS intensity. However, the formation of a complex with PD helper bacteriophages generated strong localized optical fields between the gold nanocubes and a stronger signal. Via coupling between particles, assembled nanoparticles can exhibit unique properties that are different from those of the bulk material or single nanoparticles. In this work, in particular, the interparticle coupling of gold nanocubes provided a highly concentrated local field in the nanostructure, thus providing a SERS active structure. Based on the methods described previously, well-defined assembly was difficult to achieve and fabrication procedures were complicated.

24-26

However, in the present study, we were able to fabricate readily a large

amount of well-assembled nanoparticles by simply mixing the PD bacteriophage with synthesized cube nanoparticles


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Figure 5. Phage ELISA against the PSA antigen using an anti-PSA scFv displayed at pIII. The bacteriophage displaying anti-PSA scFv was amplified using wild-type, VSGSSPDS, and PDVCSM13 helper bacteriophages. Phage ELISA results are shown as the mean ± SD of triplicate measurements. Significant differences in values between the antigen and blocking only are indicated by an asterisk. ***P < 0.001 (NS: not significant).

In summary, the purpose of this study was to develop a pVIII-engineered helper bacteriophage with optimal gold nanocube binding for use in a SERS multiplex assay. We found that the length and sequence of the exposed peptide determined the efficiency of the bacteriophage particle assembly. We enriched the phage clones by biopanning, and we selected one clone that exhibited the highest fold change after enrichment. This clone displayed Pro–Asp amino acids and amplification yields that were similar to those of the wild-type helper bacteriophages. Additionally, the clone exhibited even binding of the gold nanocubes along its length and minimal aggregation after binding. However, Raman chemical label on gold nanocubes could be more important for detecting multiple antigens. Therefore, efficient methods to conjugate various


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Raman chemicals and gold nanocubes will require further study. Simultaneously, other issues, such as the detection of differentiable signals from mixed bacteriophage remain to be discussed.

METHODS Construction of (NNK)2~6 VCSM13-pVIII randomized libraries To produce two gene fragments, we performed polymerase chain reaction (PCR), the first of which ranged from the SnaBI restriction site (nucleotides 3119–3124) at gIX in the 5′ region of gVIII to nucleotide 3217. Additionally, we introduced a KpnI site in this gene fragment using a 3′ primer encoding the KpnI restriction sequence (5′-GAAAGACAGCATCGGTACCAGGGTA GCAACGGCTAC-3′). The second gene fragment was also generated by PCR and ranged from nucleotide 3182 to the BamHI restriction site in the 3′ end of gVIII. The PCR conditions included initial denaturation at 95 °C for 5 min, 25 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. The two PCR products were subjected to 1% agarose gel electrophoresis and were purified using the QIAquick® Gel Extraction Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instructions. The purified PCR products were then subjected to an overlap extension PCR as previously described.27 After 1% agarose gel electrophoresis, the purified PCR product and the VCSM13 phagemid DNA were subjected to restriction digestion with KpnI and SnaBI following the guidelines provided by the manufacturer (New England BioLabs, Ipswich, MA, USA). The digested PCR product and the phagemid DNA were subjected to 1% agarose gel electrophoresis, purified, and subjected to ligation as previously described.19 The ligated DNA was then electroporated into Escherichia coli ER2738 electrocompetent cells (New England BioLabs) and cultured overnight as previously described.19


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To generate five NNK VCSM13-pVIII randomized libraries, PCR was performed with sense primers

(5’-GGGGTACCGATGCTGTCTTTCGCTGCTGAG(NNK)2~6GATCCCGCAAAAG

CG-3’,respectively) and anti-sense primer (5’-CAAACGAATGGATCCTCATTAAAG-3’). After restriction digestion with KpnI and BamHI, the genes were ligated to the VCSM13 phagemid DNA having KpnI restriction site. The five phagemid libraries were then individually transformed into E. coli ER2738 cells (New England BioLabs), as previously described.19 After culturing for 2 h at 37 °C with shaking, the bacterial cultures were sampled and subjected to plaque- and colony-forming assays in agar plates containing 50 µg/mL kanamycin (Duchefa Biochemie B.V., Haarlem, Netherlands) as previously described.19, 28 After each of the five library genes have been prepared, we transformed them into E. coli ER2738 cells as described above. The double-strand phagemid DNA was prepared from E. coli ER2738 cell pellets using a HiPure Plasmid Maxiprep Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions and ssDNA from phages in the culture supernatant was prepared using a Qiagen Plasmid Mini Kit (Qiagen) following the manufacturer’s supplementary protocol. Preparation of bacteriophages A single plaque was selected from the titration plate and used to infect E. coli ER2738 cells in the exponential growth phase (10 mL, OD600 = 0.8). The cells were allowed to grow with shaking at 250 rpm for 2 h at 37 °C. The culture was added to 190 mL of SB containing kanamycin (70 µg/mL) and incubated overnight at 37 °C with shaking at 250 rpm. After centrifugation at 2500 × g for 15 min, the culture was transferred to 50 mL tubes and incubated in a water bath at 70 °C for 20 min. The culture was then centrifuged at 2500 × g for 15 min. Finally, the supernatants transferred to new tubes and stored at 4 °C until used.


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Preparation of phagemid DNA A single E. coli ER2738 colony was inoculated into Super Broth (SB) medium and allowed it to grow until the optical density (OD) at 600 nm reached 0.8. A single plaque from the plate was then transferred to 2 mL of bacterial culture and incubated with shaking at 250 rpm for 2 h at 37 °C. Kanamycin was then added to a final concentration of 25 µg/mL, and the bacterial culture was continued overnight at 37 °C with shaking at 250 rpm. After centrifugation at 3000 × g for 15 min, the phagemid DNA was prepared from the bacterial pellet using a phagemid DNA preparation kit (MG Plasmid SV Mini-prep Kit, Macrogen) according to the manufacturer’s instructions. Enzyme-linked immunosorbent assay (ELISA) The 96-well plates (Coster Costar, Cambridge, MA, USA) were coated with 100 ng of recombinant PSA dissolved in coating buffer (0.1 M sodium bicarbonate, pH 8.6) overnight at 4 °C. The plates were blocked with 100 µL of 3% BSA/PSA for 1 h at 37 °C. Wild-type, VSGSSPDS, and PD-VCSM13 bacteriophages displaying anti-PSA scFv at pIII were added to individual wells in blocking buffer and incubated for 2 h at 37 °C. The plate was washed three times with 0.05% PBST, and an anti-M13-HRP antibody (GE Healthcare, Piscataway, NJ, USA) was added to the wells. After incubation for 1 h at 37 °C, the plate was washed three times with 0.05%

PBST.

Subsequently,

2,2-azinobis

[3-ethylbenzothiazoline-6-sulphonic

acid]-

diammonium salt (ABTS) substrate solution (Amresco, Solon, OH, USA) was added to each well, and the optical density was measured at 405 nm (Labsystems Multiskan Ascent microplate reader, ThermoFisher Scientific, Waltham, MA, USA). Synthesis of gold nanocubes


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Gold (III) chloride trihydrate (HAuCl4·3H2O, 99.9%), sodium borohydride (NaBH4, 99%), cetyltrimethylammonium bromide (CTAB, 99%), and L-ascorbic acid (99%) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Gold seeds were prepared by rapid reduction of HAuCl4 (10 mM, 0.25 mL) using NaBH4 in a 0.1 M aqueous CTAB solution. The seed solution was then stored at 28 °C for 3 h. The growth solution was prepared by mixing HAuCl4 (10 mM, 0.2 mL) and ascorbic acid (100 mM, 0.95 mL) with distilled water (8 mL) and CTAB (100 mM, 1.6 mL). The formation of gold nanocubes was initiated by adding 5 µL of diluted seed solution (1:10) to the growth solution. The growth solution was mixed thoroughly and left undisturbed for 15 min. The resultant particles were washed twice by centrifugation and redispersed in distilled water. Biopanning Biopanning was performed by first mixing each phage library (200 µL) with 100 µL of gold nanocube solution followed by gentle rotation for 2 h at 37 °C. Next, the gold nanocubes were centrifuged at 6000 × g for 2 min, washed with 0.1% (v/v) Tween-20 in PBS, and resuspended in solution by vortexing. To collect phage-bound gold nanocubes, the bound phages were eluted with 50 µL of elution buffer (0.2 M glycine-HCl, pH 2.2) for 10 min at 37 °C, and the eluted solution was neutralized with 5 µL of 2 M Tris-Cl (pH 9.1). The eluted phages (200 µL) were transfected into 2 mL of E. coli ER2738 cells for phage rescue, selected by growth in the presence of kanamycin, and amplified overnight. The input and output phage titers were determined as previously described. 19 Scanning electron microscopy The bacteriophage solution (2 × 109/mL, 100 µL) was mixed with 100 µL of gold nanocube solution and incubated for 1 h. The sample was prepared by dispensing droplets of the solution


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mixture onto a silicon wafer and drying rapidly. SEM images were obtained using a Zeiss Supra 55 VP scanning electron microscope (Zeiss, Oberkochen, Germany). Surface-enhanced Raman spectroscopy Gold nanocube solution (99 µL) was mixed with aqueous 0.1 mM 4-chlorobenzenethiol (4-CBT) solution for 1 h. Next, 100 µL of 4-CBT labeled gold nanocube solution was mixed with 100 µL of distilled water or bacteriophage solution. The Raman signals were then measured on a microRaman system (LabRam 300; JY-Horiba, Kyoto, Japan). The signal was collected by a 10× objective lens (0.70 NA ; Olympus, Tokyo, Japan) with back-scattering geometry equipped with a thermoelectrically cooled (–70 °C) charge-coupled device detector. The 647 nm laser line from a krypton ion laser (Innova 300C: Coherent, Santa Clara, CA, USA) was used as the excitation source. For each measurement, the acquisition time was 10 s and the sample power was about 58.8 mW at the sample.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. SEM images of the interaction with gold nanocubes and three types of phages.

AUTHOR INFORMATION Corresponding Author * Suite 509. Samsung Cancer Research Building, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. Tel.: +82 2 3668 7441; fax: +82 2 747 5769 E-mail address: [email protected] (Junho Chung)


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Present Addresses || Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea ACKNOWLEDGMENTS This research was supported by the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (NRF2012-0009555). REFERENCES [1] Kang, H., Jeong, S., Koh, Y., Cha, M. G., Yang, J. K., Kyeong, S., Kim, J., Kwak, S. Y., Chang, H. J., Lee, H., Jeong, C., Kim, J. H., Jun, B. H., Kim, Y. K., Jeong, D. H., and Lee, Y. S. (2015) Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis (vol 5, 10144, 2015), Scientific reports 5. [2] Pallaoro, A., Braun, G. B., and Moskovits, M. (2015) Biotags Based on Surface-Enhanced Raman Can Be as Bright as Fluorescence Tags, Nano Lett 15, 6745-6750. [3] Anema, J. R., Li, J. F., Yang, Z. L., Ren, B., and Tian, Z. Q. (2011) Shell-Isolated Nanoparticle-Enhanced Raman Spectroscopy: Expanding the Versatility of SurfaceEnhanced Raman Scattering, Annu Rev Anal Chem 4, 129-150. [4] Scarselli, E., Esposito, G., and Traboni, C. (1993) Receptor Phage - Display of Functional Domains of the Human High-Affinity Ige Receptor on the M13 Phage Surface, Febs Lett 329, 223-226. [5] Greenwood, J., Willis, A. E., and Perham, R. N. (1991) Multiple Display of Foreign Peptides on a Filamentous Bacteriophage - Peptides from Plasmodium-Falciparum Circumsporozoite Protein as Antigens, J Mol Biol 220, 821-827. [6] Mao, C. B., Liu, A. H., and Cao, B. R. (2009) Virus-Based Chemical and Biological Sensing, Angew Chem Int Edit 48, 6790-6810. [7] Clark, W. A., Hornelan.W, and Klein, A. G. (1962) Attempts to Freeze Some Bacteriophages to Ultralow Temperatures, Appl Microbiol 10, 463-&. [8] Jonczyk, E., Klak, M., Miedzybrodzki, R., and Gorski, A. (2011) The influence of external factors on bacteriophages-review, Folia Microbiol 56, 191-200. [9] Branston, S. D., Stanley, E. C., Ward, J. M., and Keshavarz-Moore, E. (2013) Determination of the survival of bacteriophage M13 from chemical and physical challenges to assist in its sustainable bioprocessing, Biotechnol Bioproc E 18, 560-566. [10] Huang, Y., Chiang, C. Y., Lee, S. K., Gao, Y., Hu, E. L., De Yoreo, J., and Belcher, A. M. (2005) Programmable assembly of nanoarchitectures using genetically engineered viruses, Nano Lett 5, 1429-1434. [11] Haimovic.J, Hurwitz, E., Novik, N., and Sela, M. (1970) Use of Protein-Bacteriophage Conjugates for Detection and Quantitation of Proteins, Biochim Biophys Acta 207, 125-&.


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[12] Lee, Y. J., Lee, Y., Oh, D., Chen, T., Ceder, G., and Belcher, A. M. (2010) Biologically Activated Noble Metal Alloys at the Nanoscale: For Lithium Ion Battery Anodes, Nano Lett 10, 2433-2440. [13] Essinger-Hileman, E. R., Popczun, E. J., and Schaak, R. E. (2013) Magnetic separation of colloidal nanoparticle mixtures using a material specific peptide, Chem Commun 49, 5471-5473. [14] Iannolo, G., Minenkova, O., Petruzzelli, R., and Cesareni, G. (1995) Modifying filamentous phage capsid: limits in the size of the major capsid protein, J Mol Biol 248, 835-844. [15] Dovgolevsky, E., and Haick, H. (2008) Direct Observation of the Transition Point Between Quasi-Spherical and Cubic Nanoparticles in a Two-Step Seed-Mediated Growth Method, Small 4, 2059-2066. [16] Chen, H., Sun, Z., Ni, W., Woo, K. C., Lin, H. Q., Sun, L., Yan, C., and Wang, J. (2009) Plasmon coupling in clusters composed of two-dimensionally ordered gold nanocubes, Small 5, 2111-2119. [17] Gallet, R., Kannoly, S., and Wang, I. N. (2011) Effects of bacteriophage traits on plaque formation, Bmc Microbiol 11. [18] Abedon, S. T., and Yin, J. (2009) Bacteriophage plaques: theory and analysis, Methods in molecular biology 501, 161-174. [19] Barbas, C. F. (2001) Phage display : a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [20] Catalona, W. J., Richie, J. P., Ahmann, F. R., Hudson, M. A., Scardino, P. T., Flanigan, R. C., DeKernion, J. B., Ratliff, T. L., Kavoussi, L. R., Dalkin, B. L., Waters, W. B., MacFarlane, M. T., and Southwick, P. C. (2017) Comparison of Digital Rectal Examination and Serum Prostate Specific Antigen in the Early Detection of Prostate Cancer: Results of a Multicenter Clinical Trial of 6,630 Men, J Urol 197, S200-S207. [21] Hwang, D., Yoon, A., Kim, S., Kim, H., and Chung, J. (2016) Establishment of a mammalian expression system for recombinant [-2]proPSA and a specific antibody against the truncated leader peptide, Biotechnol Appl Biochem. [22] Yang, W., Yoon, A., Lee, S., Kim, S., Han, J., and Chung, J. (2017) Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library, Exp Mol Med 49, e308. [23] Lee, H. E., Lee, H. K., Chang, H., Ahn, H. Y., Erdene, N., Lee, H. Y., Lee, Y. S., Jeong, D. H., Chung, J., and Nam, K. T. (2014) Virus templated gold nanocube chain for SERS nanoprobe, Small 10, 3007-3011. [24] Lim, II, Ip, W., Crew, E., Njoki, P. N., Mott, D., Zhong, C. J., Pan, Y., and Zhou, S. (2007) Homocysteine-mediated reactivity and assembly of gold nanoparticles, Langmuir 23, 826-833. [25] Otter, C. A., Patty, P. J., Williams, M. A., Waterland, M. R., and Telfer, S. G. (2011) Mechanically interlocked gold and silver nanoparticles using metallosupramolecular catenane chemistry, Nanoscale 3, 941-944. [26] Si, S., Raula, M., Paira, T. K., and Mandal, T. K. (2008) Reversible self-assembly of carboxylated peptide-functionalized gold nanoparticles driven by metal-ion coordination, Chemphyschem 9, 1578-1584. [27] Choi, Y. S., Yoon, S., Kim, K. L., Yoo, J., Song, P., Kim, M., Shin, Y. E., Yang, W. J., Noh, J. E., Cho, H. S., Kim, S., Chung, J., and Ryu, S. H. (2014) Computational Design of Binding Proteins to EGFR Domain II, Plos One 9.


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[28] Kramer, R. A., Cox, F., van der Horst, M., van den Oudenrijn, S., Res, P. C. M., Bia, J., Logtenberg, T., and de Kruif, J. (2003) A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein, Nucleic Acids Res 31.

TABLES Table 1. NGS sequence data derived from NNK libraries and results of colony- and plaqueforming unit assays

Library

(NNK)

(NNK)

(NNK)

(NNK)

(NNK)

a b

Theoretical complexity

3 2

1.02 x 10

4 3

3.28 x 10

6 4

1.05 x 10

7 5

3.36 x 10

9 6

1.07 x 10

Types

Phagemid DNA

Total raw sequences (%)

962149 (100)

No. of right length sequences (%) 949782 (98.7)

No. of unique clones among right length

Expected No. of b a

sequences (%)

colony

b

plaque

3

1.9 x 10 (0.2) 7

6.3 x 10

Phage (ssDNA)

884642 (100)

872544 (98.6)

2.1 x 10 (0.2)

Phagemid DNA

1179973 (100)

1154140 (97.8)

3.6 x 10 (3.1)

5

8.7 x 10

3

4 7

5.4 x10

Phage (ssDNA)

1047596 (100)

1034607 (98.7)

3.7 x 10 (3.6)

Phagemid DNA

1211710 (100)

1074929 (88.7)

5.2 x 10 (48.4)

5

2.0 x 10

4

5 7

1204805 (100)

1087267 (90.2)

5.4 x 10 (49.7)

Phagemid DNA

1001148 (100)

700256 (69.9)

6.4 x 10 (91.4)

5

1.2 x 10

5.9 x10

Phage (ssDNA)

5

5 7

6.5 x10

Phage (ssDNA)

1196370 (100)

819030 (68.5)

7.3 x 10 (89.1)

Phagemid DNA

1221895 (100)

879703 (72.0)

7.7 x 10 (87.5)

Phage (ssDNA)

Expected No. of

5

< 10

5

5 8

3.9 x10 1343694 (100)

1196378 (89.0)

5

< 10

6

1.0 x 10 (83.6)

The fraction of unique clones among the clones with right length sequence. The number of transformants was calculated by multiplying the number of colonies or plaques by the culture volume and dividing by the plating volume.


21


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Screening of Pro–Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes

Hwa Kyoung Lee, Yujean Lee, Hyori Kim, Hye-Eun Lee, Hyejin Chang, Ki Tae Nam, Dae Hong Jeong, Junho Chung

For Table of Contents Use only


1

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


SnaB I

BamH I

Kpn I

VCSM13 genome

gVIII Leader peptide

gIII exposed

α-helix

MKKSLVLKASVAVATLVPMLSFAAEGDDPAKAA……KKFTSKAS (NNK)6 (NNK)5 (NNK)4 (NNK)3 (NNK)2


Wild type pVIII sequence


1 2 3 4 5 6 7 8 9 110 00% 11 12 13 148 0 % 15 16 176 0 % 18 19 20 214 0 % 22 23 242 0 % 25 26 27 28 0 % 29 30 31 32 33 34 35 36 37 38 39 40 41 42

(N N K ) 2

6.89

Library

93.33

Selected

VP RD DS SN PD PV LH LL VH SD YD SY QG PN WN SP DT

Page 24 of 30

(N N K )3 100%

NVS ELS SDG Q LD MSS IE A RRA D ID DVS ESQ ENS RMG DNA EVP

80% 60% 40% 20% 0%

(N N K )4

Library

Selected


100% 80% 60% 40% AG DS EGGD GEGE GGSE

20% 0%

Library

Selected

Page 25 of 30

B

A

**

D

C

1cm

1cm

1

1cm

**

E

**

3

**

x 1011 pFu/ml

0.8

mm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


0.6 0.4 0.2

2

1

0

0 Wild-type

VSGSSPDS

PD

Wild-type


VSGSSPDS

PD


E

A

H

G

200nm

B

F

200nm

I 14000 14000

200nm

D

1083 1100 1063

12000 12000

Intensity (a.u.)

C

200nm 200nm

200nm

200nm

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 26 of 30

1177

10000 541 10000

740

8000 800 6000 600 600

1μm

200nm


600

800

800

1000

1200

1000 -1 1200

Raman ) RamanShift Shift(cm (cm-1)

Page 27 of 30

NS 1.8

Absorbance at 650nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


***

***

1.5 1.2

***

0.9 0.6 0.3 0.0

Wild-type PSA(Antigen)

VSGSSPDS

PD

Blocking only



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


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